Vesicular stomatitis viruses

ABSTRACT

This document provides methods and materials related to vesicular stomatitis viruses. For example, vesicular stomatitis viruses, nucleic acid molecules encoding VSV polypeptides, methods for making vesicular stomatitis viruses, and methods for using vesicular stomatitis viruses to treat cancer are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/925,279, filed Mar. 19, 2018, which is a continuation of U.S.application Ser. No. 15/246,189, filed Aug. 24, 2016 (now U.S. Pat. No.9,951,117), which is a divisional of U.S. application Ser. No.13/820,453 (now U.S. Pat. No. 9,428,736), filed May 20, 2013, which is aNational Stage Application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2011/050227, filed Sep. 1, 2011, which claims thebenefit of U.S. Provisional Application No. 61/379,644, filed Sep. 2,2010. The contents of the foregoing application are hereby incorporatedby reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberCA129966 awarded by the National Institute of Health. The government hascertain rights in the invention.

1. TECHNICAL FIELD

This document relates to methods and materials involved in making andusing vesicular stomatitis viruses. For example, this document relatesto vesicular stomatitis viruses, nucleic acid molecules, methods formaking vesicular stomatitis viruses, and methods for using vesicularstomatitis viruses to treat cancer.

2. BACKGROUND INFORMATION

Vesicular stomatitis virus (VSV) is a member of the Rhabdoviridaefamily. The VSV genome is a single molecule of negative-sense RNA thatencodes five major polypeptides: a nucleocapsid (N) polypeptide, aphosphoprotein (P) polypeptide, a matrix (M) polypeptide, a glycoprotein(G) polypeptide, and a viral polymerase (L) polypeptide.

SUMMARY

This document provides methods and materials related to vesicularstomatitis viruses. For example, this document provides vesicularstomatitis viruses, nucleic acid molecules encoding VSV polypeptides,methods for making vesicular stomatitis viruses, and methods for usingvesicular stomatitis viruses to treat cancer.

As described herein, vesicular stomatitis viruses can be designed tohave a nucleic acid molecule that encodes a VSV N polypeptide, a VSV Ppolypeptide, a VSV M polypeptide, a VSV G polypeptide, a VSV Lpolypeptide, an interferon (IFN) polypeptide (e.g., a human IFN-βpolypeptide), and a sodium iodide symporter (NIS) polypeptide (e.g., ahuman NIS polypeptide). The nucleic acid encoding the IFN polypeptidecan be positioned between the nucleic acid encoding the VSV Mpolypeptide and the nucleic acid encoding the VSV G polypeptide. Such aposition can allow the viruses to express an amount of the IFNpolypeptide that is effective to activate anti-viral innate immuneresponses in non-cancerous tissues, and thus alleviate potential viraltoxicity, without impeding efficient viral replication in cancer cells.The nucleic acid encoding the NIS polypeptide can be positioned betweenthe nucleic acid encoding the VSV G polypeptide and the VSV Lpolypeptide. Such a position of can allow the viruses to express anamount of the NIS polypeptide that (a) is effective to allow selectiveaccumulation of iodide in infected cells, thereby allowing both imagingof viral distribution using radioisotopes and radiotherapy targeted toinfected cancer cells, and (b) is not so high as to be toxic to infectedcells. Positioning the nucleic acid encoding an IFN polypeptide betweenthe nucleic acid encoding the VSV M polypeptide and the nucleic acidencoding the VSV G polypeptide and positioning the nucleic acid encodinga NIS polypeptide between the nucleic acid encoding the VSV Gpolypeptide and the VSV L polypeptide within the genome of a vesicularstomatitis virus can result in vesicular stomatitis viruses that areviable, that have the ability to replicate and spread, that expressappropriate levels of functional IFN polypeptides, and that expressionappropriate levels of functional NIS polypeptides to take upradio-iodine for both imaging and radio-virotherapy.

In some cases, this document features a vesicular stomatitis viruscomprising an RNA molecule. The RNA molecule comprises, or consistsessentially of, in a 3′ to 5′ direction, a nucleic acid sequence that isa template for a positive sense transcript encoding a VSV N polypeptide,a nucleic acid sequence that is a template for a positive sensetranscript encoding a VSV P polypeptide, a nucleic acid sequence that isa template for a positive sense transcript encoding a VSV M polypeptide,a nucleic acid sequence that is a template for a positive sensetranscript encoding an IFN polypeptide, a nucleic acid sequence that isa template for a positive sense transcript encoding a VSV G polypeptide,a nucleic acid sequence that is a template for a positive sensetranscript encoding a NIS polypeptide, and a nucleic acid sequence thatis a template for a positive sense transcript encoding a VSV Lpolypeptide. The IFN polypeptide can be a human IFN beta polypeptide.The NIS polypeptide can be a human NIS polypeptide. The virus canexpress the IFN polypeptide when the virus infects a mammalian cell. Thevirus can express the NIS polypeptide when the virus infects a mammaliancell.

In another aspect, this document features a composition comprising, orconsisting essentially of, a vesicular stomatitis virus comprising RNAmolecule. The RNA molecule comprises, or consists essentially of, in a3′ to 5′ direction, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV N polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding aVSV P polypeptide, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV M polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding anIFN polypeptide, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV G polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding aNIS polypeptide, and a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV L polypeptide. The IFNpolypeptide can be a human IFN beta polypeptide. The NIS polypeptide canbe a human NIS polypeptide. The virus can express the IFN polypeptidewhen the virus infects a mammalian cell. The virus can express the NISpolypeptide when the virus infects a mammalian cell.

In another aspect, this document features a nucleic acid moleculecomprising a nucleic acid strand comprising, or consisting essentiallyof, in a 3′ to 5′ direction, a nucleic acid sequence that is a templatefor a positive sense transcript encoding a VSV N polypeptide, a nucleicacid sequence that is a template for a positive sense transcriptencoding a VSV P polypeptide, a nucleic acid sequence that is a templatefor a positive sense transcript encoding a VSV M polypeptide, a nucleicacid sequence that is a template for a positive sense transcriptencoding an IFN polypeptide, a nucleic acid sequence that is a templatefor a positive sense transcript encoding a VSV G polypeptide, a nucleicacid sequence that is a template for a positive sense transcriptencoding a NIS polypeptide, and a nucleic acid sequence that is atemplate for a positive sense transcript encoding a VSV L polypeptide.The IFN polypeptide can be a human IFN beta polypeptide. The NISpolypeptide can be a human NIS polypeptide.

In another aspect, this document features a method for treating cancer.The method comprises, or consists essentially of, administering acomposition comprising vesicular stomatitis viruses to a mammalcomprising cancer cells. The vesicular stomatitis viruses comprise anRNA molecule comprising, or consisting essentially of, in a 3′ to 5′direction, a nucleic acid sequence that is a template for a positivesense transcript encoding a VSV N polypeptide, a nucleic acid sequencethat is a template for a positive sense transcript encoding a VSV Ppolypeptide, a nucleic acid sequence that is a template for a positivesense transcript encoding a VSV M polypeptide, a nucleic acid sequencethat is a template for a positive sense transcript encoding an IFNpolypeptide, a nucleic acid sequence that is a template for a positivesense transcript encoding a VSV G polypeptide, a nucleic acid sequencethat is a template for a positive sense transcript encoding a NISpolypeptide, and a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV L polypeptide, whereinadministration of the composition to the mammal is under conditionswherein the vesicular stomatitis viruses infect the cancer cells to forminfected cancer cells, wherein the infected cancer cells express the IFNpolypeptide and the NIS polypeptide, and wherein the number of cancercells within the mammal is reduced following the administration. Themammal can be a human. The IFN polypeptide can be a human IFN betapolypeptide. The MS polypeptide can be a human NIS polypeptide.

In another aspect, this document features a method for inducing tumorregression in a mammal. The method comprises, or consists essentiallyof, administering a composition comprising vesicular stomatitis virusesto a mammal comprising a tumor. The vesicular stomatitis virusescomprises an RNA molecule comprising, or consisting essentially of, in a3′ to 5′ direction, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV N polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding aVSV P polypeptide, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV M polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding anIFN polypeptide, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV G polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding aNIS polypeptide, and a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV L polypeptide, whereinadministration of the composition to the mammal is under conditionswherein the vesicular stomatitis viruses infect tumor cells of the tumorto form infected tumor cells, wherein the infected tumor cells expressthe IFN polypeptide and the NIS polypeptide. The mammal can be a human.The IFN polypeptide can be a human IFN beta polypeptide. The NISpolypeptide can be a human NIS polypeptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1F. FIG. 1A is a schematic diagram of the genome arrangement ofan exemplary vesicular stomatitis virus containing nucleic acid encodingan IFN polypeptide (e.g., a human or mouse IFNβ polypeptide) and nucleicacid encoding a NIS polypeptide (e.g., a human NIS polypeptide). FIG. 1Bis a graph plotting the viral titer of vesicular stomatitis virusescontaining nucleic acid encoding a green fluorescent protein (GFP)polypeptide (VSV-GFP; (•)), VSV-mIFN-NIS (▪), or VSV-hIFN-NIS (♦)determined using BHK cells infected at MOI 1.0. FIG. 1C is a graphplotting radio-iodide uptake of cells infected with VSV-mIFN-NIS orVSV-hIFN-NIS in the presence or absence of KCLO4, a NIS inhibitor(+inh.). VSV-GFP was used as a control. FIG. 1D contains bar graphsplotting the level of secretion of murine or human IFNβ measured byELISA from mock infected cells or cells infected with VSV-mIFN-NIS (VmN)or VSV-hIFN-NIS (VhN). FIG. 1E contains bar graphs plotting IFNresponsiveness of 5TGM1 and MPC-11 murine myeloma cells compared to B-16murine melanoma cells as assessed by pre-treating cells with 100 U/mLmurine IFNβ for 12 hours, followed by infection with VSV-GFP (MOI 1.0).FIG. 1F contains graphs plotting proliferation of viable cells resultsassessed by an MTT assay at 48 hours post-infection (plotted as % ofuntreated cells). 5TGM1 and MPC-11 oncolysis was monitored followinginfection with VSV-mIFN-NIS or VSV-hIFN-NIS (MOI 1.0) by measuring cellviability at 12 hour intervals by MTT assay.

FIGS. 2A and 2B. FIG. 2A is a graph plotting the level of IFNpolypeptide release from BHK cells infected with either VSV-mIFN-NIS orVSV-mIFN and monitored over a 48 hour time period. The mouse IFNpolypeptide levels were measured in the supernatant using an ELISAdesigned to detect mouse IFN polypeptides expression levels. FIG. 2B isa graph plotting the level of 1-125 uptake by BHK cells infected witheither VSV-mIFN-NIS or VSV-mIFN at the indicated time (hours).

FIGS. 3A-3C contain results from monitoring intratumoral spread ofintravenously administered VSV-IFN-NIS. Female, 6-10 week oldC57Bl6/KaLwRij mice bearing subcutaneous syngeneic 5TGM1 myeloma tumorswere treated with a single intravenous (IV) dose of 100 μL PBS (control)or 1×10⁸ TCID50VSV-mIFN-NIS. (A) SPECT-CT imaging was carried out at 24hour intervals post-treatment following administration with 0.5 mCiTc-99m. Tumor specific Tc-99m uptake was quantified in PBS treated mice(n=2) and VSV-mIFN-NIS treated mice (n=5). (B) Intratumoral viraldistribution was monitored by harvesting tumors following SPECT-CTimaging and corollary analysis of adjacent tumor section byautoradiography, and IF was performed. IF was used to detect VSVantigens (which stained red) and cells undergoing cell death by TUNELstaining (which stained green) at 24 hour time periods. (C) IntratumoralVSV and TUNEL were quantified using from 4 images from n=3 tumors (n=2at 72 hours) using ImageJ software and shown as a percentage of tumorarea. There was a significant increase in both VSV(+) and TUNEL(+)between 24 and 48 hours post treatment using t-test (P=0.0455 andP=0.0163, respectively).

FIGS. 4A-4D contain results of intratumoral viral entry, spread, andoncolysis following intravenous delivery. (A) 5TGM1 tumors wereharvested and analyzed by IF at (i) 6 hours, (ii) 12 hours, (iii) 18hours, and (iv-vi) 24 hours following intravenous VSV-mIFN-NISadministration indicating VSV infected cells (which stained green) andtumor blood vessels by CD31 staining (which stained red). Magnification100×. (B) High magnification view of treated tumors showing intact tumorblood vessels (which stained red) in proximity of (i) VSV infected tumorcells (which stained green) and (ii) TUNEL positive cells undergoingcells death (which stained green) with Hoescht stained nuclei (whichstained blue). (C) Intratumoral foci (n=8) from tumors harvested at 6hour intervals were measured using ImageJ software, and average diameterwas plotted over time. Significance of diameter growth was measured byt-test, and P values are shown along the top of the graph. Diameterswere used to measure average foci volume over time. (D) Images of tumorat 48 hours post VSV-mIFN-NIS treatment were used to quantify viable rimof infected cells to obtain an average of −10 cells being infected ateach round of infection prior to cell death.

FIG. 5 contains a set of three images of a tumor from the same mouse,undergoing whole body SPECT-CT imaging to monitor viral distribution(top, left), tumor autoradiography to show specific regions ofradio-isotope uptake (top, right), and anti-VSV and DAPI staining toshow that regions of NIS expression correspond to intratumoral VSVstaining (bottom).

FIGS. 6A-6C contain results demonstrating a potent therapeutic efficacyof systemically administered VSV-IFN-NIS. Mice bearing subcutaneous5TGM1 tumors were treated with a single intravenous dose of PBS,VSV-mIFN-NIS, or VSV-hIFN-NIS. (A) Tumor burden was measured by serialcaliper measurements to calculate tumor volume over time. (B) Tumorresponses were categorized into tumor progression, regression, orregression with relapse. Statistical difference in incidence of relapseas proportion of mice with tumor regression was measured Fischer Exacttest indicating significantly higher rate of tumor relapse inVSV-hIFN-NIS treated mice vs. VSV-mIFN-NIS treated mice (P=0.009). (C)Generation of VSV neutralizing antibodies was measured in serum of n=2(PBS treated) and n=3 (VSV-IFN-NIS treated) mice in the first 5 dayspost treatment and plotted as the minimum fold dilution that protectsfrom infection with 500 TCID50 VSV.

FIGS. 7A-7E contain results demonstrating that immune mediatedelimination of tumor cells prevents tumor relapse. (A) Quantification ofmurine IFNβ in serum of mice bearing subcutaneous 5TGM1 tumors treatedintravenously with PBS, VSV-mIFN-NIS, or VSV-hIFN-NIS measured by ELISA.(B) Mice that had complete tumor regression in response to VSV-mIFN-NIStreatment and naïve age-matched syngeneic mice (n=6 each) werechallenged with 1×10⁷ STGM1 cells subcutaneously, and tumor occurrenceby day 21 post challenge is shown. (C) Immunotherapeutic efficacy ofsingle dose VSV-infected 5TGM1 cells administered subcutaneously (1×10⁷STGM1 cells infected with VSV-mIFN-NIS at MOI 10 implanted on leftflank) at 1 day post or 5 days prior to tumor implantation (5×10⁶subcutaneous 5TGM1 cells on right flank). Log rank survival analysiscomparison revealed day (−5) vaccination prolongs survival of micefollowing tumor implantation compared to unvaccinated mice (P=0.0253).(D) Mice bearing subcutaneous 5TGM1 tumors were treated with singleintravenous dose of (i) PBS, (ii) VSV-mIFN-NIS, or (iii) VSV-mIFN-NIS inthe presence or absence of antibodies to deplete CD4⁺ or CD8⁺ T cells.Tumor burden was measured by serial caliper measurements. (E) Tumorresponses for the mice described in FIG. 7D were categorized intoprogression, regression, or regression+relapse. Relapse rates werecompared by Fischer Exact test indicating that VSV-mIFN-NIS+T-celldepletion exhibited a higher rate of tumor relapse compared toVSV-mIFN-NIS treatment alone (P=0.0498).

FIGS. 8A and 8B are detailed immunofluorescence images of intratumoralfoci of infection. A 5TGM1 tumor was harvested 24 hours post intravenousVSV-mIFN-NIS injection, frozen in OCT, and sectioned. IF was performedto detect VSV (which stained red), dying cells by TUNEL staining (whichstained green), and tumor cell nuclei by Hoescht staining (which stainedblue). Distinct roughly spherical intratumoral foci of VSV infectioncontain a central region of VSV infected cells undergoing cell death anda rim of infected, viable cells in the periphery.

FIGS. 9A-9B contain images demonstrating that functional NIS activity islimited to regions of VSV infected viable cells. 5TGM1 tumors wereharvested at 48 hours (A) or 72 hours (B) post intravenous VSV-IFN-NISinjection and sectioned. Adjacent sections were subject to (i)autoradiography, (ii) IF shown at 20× magnification, and (iii) IF shownat 100× magnification to detect VSV (which stained red) and dying cellsby TUNEL staining (which stained green).

FIG. 10 is a diagram of exemplary intragenic regions of vesicularstomatitis viruses that contain inserted transgenes. The transgenes areflanked by viral start and stop sequences involved in transcription. TheIFN nucleic acid is inserted into a NotI cloning site engineered betweenthe VSV M and G nucleic acid sequences. The NIS nucleic acid is insertedinto XhoI and NheI restriction sites engineered between the VSV G and Lnucleic acid sequences.

DETAILED DESCRIPTION

This document provides methods and materials related to vesicularstomatitis viruses. For example, this document provides vesicularstomatitis viruses, nucleic acid molecules encoding VSV polypeptides,methods for making vesicular stomatitis viruses, and methods for usingvesicular stomatitis viruses to treat cancer.

As described herein, a vesicular stomatitis virus can be designed tohave a nucleic acid molecule that encodes a VSV N polypeptide, a VSV Ppolypeptide, a VSV M polypeptide, a VSV G polypeptide, a VSV Lpolypeptide, an IFN polypeptide, and a NIS polypeptide. It will beappreciated that the sequences described herein with respect to avesicular stomatitis virus are incorporated into a plasmid coding forthe positive sense cDNA of the viral genome allowing generation of thenegative sense genome of vesicular stomatitis viruses. Thus, it will beappreciated that a nucleic acid sequence that encodes a VSV polypeptide,for example, can refer to an RNA sequence that is the template for thepositive sense transcript that encodes (e.g., via direct translation)that polypeptide.

The nucleic acid encoding the IFN polypeptide can be positioneddownstream of the nucleic acid encoding the VSV M polypeptide (FIG. 1A).For example, nucleic acid encoding the IFN polypeptide can be positionedbetween the nucleic acid encoding the VSV M polypeptide and the nucleicacid encoding the VSV G polypeptide. Such a position can allow theviruses to express an amount of the IFN polypeptide that is effective toactivate anti-viral innate immune responses in non-cancerous tissues,and thus alleviate potential viral toxicity, without impeding efficientviral replication in cancer cells.

Any appropriate nucleic acid encoding an IFN polypeptide can be insertedinto the genome of a vesicular stomatitis virus. For example, nucleicacid encoding an IFN beta polypeptide can be inserted into the genome ofa vesicular stomatitis virus. Examples of nucleic acid encoding IFN betapolypeptides that can be inserted into the genome of a vesicularstomatitis virus include, without limitation, nucleic acid encoding ahuman IFN beta polypeptide of the nucleic acid sequence set forth inGenBank® Accession No. NM_002176.2 (GI No. 50593016), nucleic acidencoding a mouse IFN beta polypeptide of the nucleic acid sequence setforth in GenBank® Accession Nos. NM_010510.1 (GI No. 6754303),BC119395.1 (GI No. 111601321), or BC119397.1 (GI No. 111601034), andnucleic acid encoding a rat IFN beta polypeptide of the nucleic acidsequence set forth in GenBank® Accession No. NM_019127.1 (GI No.9506800).

The nucleic acid encoding the NIS polypeptide can be positioneddownstream of the nucleic acid encoding the VSV G polypeptide (FIG. 1A).For example, nucleic acid encoding the NIS polypeptide can be positionedbetween the nucleic acid encoding the VSV G polypeptide and the nucleicacid encoding the VSV L polypeptide. Such a position of can allow theviruses to express an amount of the NIS polypeptide that (a) iseffective to allow selective accumulation of iodide in infected cells,thereby allowing both imaging of viral distribution using radioisotopesand radiotherapy targeted to infected cancer cells, and (b) is not sohigh as to be toxic to infected cells.

Any appropriate nucleic acid encoding a NIS polypeptide can be insertedinto the genome of a vesicular stomatitis virus. For example, nucleicacid encoding a human NIS polypeptide can be inserted into the genome ofa vesicular stomatitis virus. Examples of nucleic acid encoding NISpolypeptides that can be inserted into the genome of a vesicularstomatitis virus include, without limitation, nucleic acid encoding ahuman NIS polypeptide of the nucleic acid sequence set forth in GenBank®Accession Nos. NM_000453.2 (GI No. 164663746), BC105049.1 (GI No.85397913), or BC105047.1 (GI No. 85397519), nucleic acid encoding amouse NIS polypeptide of the nucleic acid sequence set forth in GenBank®Accession Nos. NM_053248.2 (GI No. 162138896), AF380353.1 (GI No.14290144), or AF235001.1 (GI No. 12642413), nucleic acid encoding achimpanzee NIS polypeptide of the nucleic acid sequence set forth inGenBank® Accession No. XM_524154 (GI No. 114676080), nucleic acidencoding a dog NIS polypeptide of the nucleic acid sequence set forth inGenBank® Accession No. XM_541946 (GI No. 73986161), nucleic acidencoding a cow NIS polypeptide of the nucleic acid sequence set forth inGenBank® Accession No. XM_581578 (GI No. 297466916), nucleic acidencoding a pig NIS polypeptide of the nucleic acid sequence set forth inGenBank® Accession No. NM_214410 (GI No. 47523871), and nucleic acidencoding a rat NIS polypeptide of the nucleic acid sequence set forth inGenBank® Accession No. NM_052983 (GI No. 158138504).

Nucleic acid inserted into the genome of a vesicular stomatitis virus(e.g., nucleic acid encoding a NIS polypeptide and/or nucleic acidencoding an IFN polypeptide) can be flanked by viral intragenic regionscontaining the gene transcription start and stop codes required fortranscription of the inserted nucleic acid sequences by the viralpolymerase. Examples of such viral intragenic regions include, withoutlimitation, those set forth in FIG. 10.

The nucleic acid sequences of a vesicular stomatitis virus providedherein that encode a VSV N polypeptide, a VSV P polypeptide, a VSV Mpolypeptide, a VSV G polypeptide, and a VSV L polypeptide can be from aVSV Indiana strain as set forth in GenBank® Accession Nos. NC_001560 (GINo. 9627229) or can be from a VSV New Jersey strain.

In one aspect, this document provides vesicular stomatitis virusescontaining a nucleic acid molecule (e.g., an RNA molecule) having, in a3′ to 5′ direction, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV N polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding aVSV P polypeptide, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV M polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding anIFN polypeptide, a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV G polypeptide, a nucleic acidsequence that is a template for a positive sense transcript encoding aNIS polypeptide, and a nucleic acid sequence that is a template for apositive sense transcript encoding a VSV L polypeptide. Such vesicularstomatitis viruses can infect cells (e.g., cancer cells) and direct theexpression of the IFN polypeptide and the NIS polypeptide by theinfected cells.

Any appropriate method can be used to insert nucleic acid (e.g., nucleicacid encoding an IFN polypeptide and/or nucleic acid encoding a NISpolypeptide) into the genome of a vesicular stomatitis virus. Forexample, the methods described elsewhere (Obuchi et al., J. Virol.,77(16):8843-56 (2003)); Goel et al., Blood, 110(7):2342-50 (2007)); andKelly et al., J. Virol., 84(3):1550-62 (2010)) can be used to insertnucleic acid into the genome of a vesicular stomatitis virus. Anyappropriate method can be used to identify vesicular stomatitis virusescontaining a nucleic acid molecule described herein. Such methodsinclude, without limitation, PCR and nucleic acid hybridizationtechniques such as Northern and Southern analysis. In some cases,immunohistochemistry and biochemical techniques can be used to determineif a vesicular stomatitis virus contains a particular nucleic acidmolecule by detecting the expression of a polypeptide encoded by thatparticular nucleic acid molecule.

In another aspect, this document provides nucleic acid molecules thatencode a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, anIFN polypeptide, a VSV G polypeptide, a NIS polypeptide, and a VSV Lpolypeptide. For example, a nucleic acid molecule provided herein can bea single nucleic acid molecule that includes a nucleic acid sequencethat encodes a VSV N polypeptide, a nucleic acid sequence that encodes aVSV P polypeptide, a nucleic acid sequence that encodes a VSV Mpolypeptide, a nucleic acid sequence that encodes an IFN polypeptide, anucleic acid sequence that encodes a VSV G polypeptide, a nucleic acidsequence that encodes a NIS polypeptide, and a nucleic acid sequencethat encodes a VSV L polypeptide.

The term “nucleic acid” as used herein encompasses both RNA (e.g., viralRNA) and DNA, including cDNA, genomic DNA, and synthetic (e.g.,chemically synthesized) DNA. A nucleic acid can be double-stranded orsingle-stranded. A single-stranded nucleic acid can be the sense strandor the antisense strand. In addition, a nucleic acid can be circular orlinear.

This document also provides method for treating cancer (e.g., to reducetumor size, inhibit tumor growth, or reduce the number of viable tumorcells). For example, a vesicular stomatitis virus provided herein can beadministered to a mammal having cancer to reduce tumor size, to inhibitcancer cell or tumor growth, and/or to reduce the number of viablecancer cells within the mammal. A vesicular stomatitis virus providedherein can be propagated in host cells in order to increase theavailable number of copies of that virus, typically by at least 2-fold(e.g., by 5- to 10-fold, by 50- to 100-fold, by 500- to 1,000-fold, oreven by as much as 5,000- to 10,000-fold). In some cases, a vesicularstomatitis virus provided herein can be expanded until a desiredconcentration is obtained in standard cell culture media (e.g., DMEM orRPMI-1640 supplemented with 5-10% fetal bovine serum at 37° C. in 5%CO₂). A viral titer typically is assayed by inoculating cells (e.g.,BHK-21 cells) in culture.

Vesicular stomatitis viruses provided herein can be administered to acancer patient by, for example, direct injection into a group of cancercells (e.g., a tumor) or intravenous delivery to cancer cells. Avesicular stomatitis virus provided herein can be used to treatdifferent types of cancer including, without limitation, myeloma (e.g.,multiple myeloma), melanoma, glioma, lymphoma, mesothelioma, and cancersof the lung, brain, stomach, colon, rectum, kidney, prostate, ovary,breast, pancreas, liver, and head and neck.

Vesicular stomatitis viruses provided herein can be administered to apatient in a biologically compatible solution or a pharmaceuticallyacceptable delivery vehicle, by administration either directly into agroup of cancer cells (e.g., intratumorally) or systemically (e.g.,intravenously). Suitable pharmaceutical formulations depend in part uponthe use and the route of entry, e.g., transdermal or by injection. Suchforms should not prevent the composition or formulation from reaching atarget cell (i.e., a cell to which the virus is desired to be deliveredto) or from exerting its effect. For example, pharmacologicalcompositions injected into the blood stream should be soluble.

While dosages administered will vary from patient to patient (e.g.,depending upon the size of a tumor), an effective dose can be determinedby setting as a lower limit the concentration of virus proven to be safeand escalating to higher doses of up to 10¹² pfu, while monitoring for areduction in cancer cell growth along with the presence of anydeleterious side effects. A therapeutically effective dose typicallyprovides at least a 10% reduction in the number of cancer cells or intumor size. Escalating dose studies can be used to obtain a desiredeffect for a given viral treatment (see, e.g., Nies and Spielberg,“Principles of Therapeutics,” In Goodman & Gilman's The PharmacologicalBasis of Therapeutics, eds. Hardman, et al., McGraw-Hill, N Y, 1996, pp43-62).

Vesicular stomatitis viruses provided herein can be delivered in a doseranging from, for example, about 10³ pfu to about 10¹² pfu (e.g., about10⁵ pfu to about 10¹² pfu, about 10⁶ pfu to about 10¹¹ pfu, or about 10⁶pfu to about 10¹⁰ pfu). A therapeutically effective dose can be providedin repeated doses. Repeat dosing is appropriate in cases in whichobservations of clinical symptoms or tumor size or monitoring assaysindicate either that a group of cancer cells or tumor has stoppedshrinking or that the degree of viral activity is declining while thetumor is still present. Repeat doses can be administered by the sameroute as initially used or by another route. A therapeutically effectivedose can be delivered in several discrete doses (e.g., days or weeksapart) and in one embodiment, one to about twelve doses are provided.Alternatively, a therapeutically effective dose of vesicular stomatitisviruses provided herein can be delivered by a sustained releaseformulation. In some cases, a vesicular stomatitis virus provided hereincan be delivered in combination with pharmacological agents thatfacilitate viral replication and spread within cancer cells or agentsthat protect non-cancer cells from viral toxicity. Examples of suchagents are described elsewhere (Alvarez-Breckenridge et al., Chem. Rev.,109(7):3125-40 (2009)).

Vesicular stomatitis viruses provided herein can be administered using adevice for providing sustained release. A formulation for sustainedrelease of vesicular stomatitis viruses can include, for example, apolymeric excipient (e.g., a swellable or non-swellable gel, orcollagen). A therapeutically effective dose of vesicular stomatitisviruses can be provided within a polymeric excipient, wherein theexcipient/virus composition is implanted at a site of cancer cells(e.g., in proximity to or within a tumor). The action of body fluidsgradually dissolves the excipient and continuously releases theeffective dose of virus over a period of time. Alternatively, asustained release device can contain a series of alternating active andspacer layers. Each active layer of such a device typically contains adose of virus embedded in excipient, while each spacer layer containsonly excipient or low concentrations of virus (i.e., lower than theeffective dose). As each successive layer of the device dissolves,pulsed doses of virus are delivered. The size/formulation of the spacerlayers determines the time interval between doses and is optimizedaccording to the therapeutic regimen being used.

Vesicular stomatitis viruses provided herein can be directlyadministered. For example, a virus can be injected directly into a tumor(e.g., a breast cancer tumor) that is palpable through the skin.Ultrasound guidance also can be used in such a method. Alternatively,direct administration of a virus can be achieved via a catheter line orother medical access device, and can be used in conjunction with animaging system to localize a group of cancer cells. By this method, animplantable dosing device typically is placed in proximity to a group ofcancer cells using a guidewire inserted into the medical access device.An effective dose of a vesicular stomatitis virus provided herein can bedirectly administered to a group of cancer cells that is visible in anexposed surgical field.

In some cases, vesicular stomatitis viruses provided herein can bedelivered systemically. For example, systemic delivery can be achievedintravenously via injection or via an intravenous delivery devicedesigned for administration of multiple doses of a medicament. Suchdevices include, but are not limited to, winged infusion needles,peripheral intravenous catheters, midline catheters, peripherallyinserted central catheters, and surgically placed catheters or ports.

The course of therapy with a vesicular stomatitis virus provided hereincan be monitored by evaluating changes in clinical symptoms or by directmonitoring of the number of cancer cells or size of a tumor. For a solidtumor, the effectiveness of virus treatment can be assessed by measuringthe size or weight of the tumor before and after treatment. Tumor sizecan be measured either directly (e.g., using calipers), or by usingimaging techniques (e.g., X-ray, magnetic resonance imaging, orcomputerized tomography) or from the assessment of non-imaging opticaldata (e.g., spectral data). For a group of cancer cells (e.g., leukemiacells), the effectiveness of viral treatment can be determined bymeasuring the absolute number of leukemia cells in the circulation of apatient before and after treatment. The effectiveness of viral treatmentalso can be assessed by monitoring the levels of a cancer specificantigen. Cancer specific antigens include, for example, carcinoembryonicantigen (CEA), prostate specific antigen (PSA), prostatic acidphosphatase (PAP), CA 125, alpha-fetoprotein (AFP), carbohydrate antigen15-3, and carbohydrate antigen 19-4.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Single Dose Intravenous Virotherapy Using VesicularStomatitis Viruses that Express an IFN Polypeptide and/or a NISPolypeptide Mediates Oncolytic Tumor Debulking and ImmunotherapeuticEradication of Residual Disease

Vesicular stomatitis viruses designed to express a mouse IFN betapolypeptide (VSV-mIFN) and vesicular stomatitis viruses designed toexpress both a mouse IFN beta polypeptide and a human NIS polypeptide(VSV-mIFN-NIS; FIG. 1A) were created using methods similar to thosedescribed elsewhere (Obuchi et al., J. Virol., 77(16):8843-56 (2003));Goel et al., Blood, 110(7):2342-50 (2007)); Kelly et al., J. Virol.,84(3):1550-62 (2010); and Lawson et al., Proc. Nat'l. Acad. Sci. USA,92(10):4477-81 (1995) Erratum in: Proc. Nat'l. Acad. Sci. USA,92(19):9009 (1995)). Likewise, vesicular stomatitis viruses designed toexpress a human IFN beta polypeptide and a human NIS polypeptide(VSV-hIFN-NIS; FIG. 1A) were created. Briefly, nucleic acid sequences ofdesired transgenes were generated with specific restriction sites usingPCR. The transgenes were inserted at specific insertion sites into aplasmid encoding the positive strand of the VSV genome in a 5′ to 3′orientation. The modified plasmid was expanded and infective virus wasrecovered by infection with vaccinia virus coding for required T7polymerase and transfection of VSV viral proteins N, P, and L. Thisallowed production of required viral polypeptides allowing generation ofthe negative sense viral genome that was assembled into infectivevirions. Recovered virus was amplified, and infective dose was measuredon an appropriate cell line in culture (e.g., BHK-21 cells). The nucleicacid sequence of the mouse IFN beta polypeptide used to make thesevesicular stomatitis viruses is set forth in GenBank® Accession No.NM_010510.1 (GI No. 6754303). The nucleic acid sequence of the human IFNbeta polypeptide used to make these vesicular stomatitis viruses is setforth in GenBank® Accession No. NM_002176.2 (GI No. 50593016). Thenucleic acid sequence of the human NIS polypeptide used to make thesevesicular stomatitis viruses is set forth in GenBank® Accession No.NM_000453.2 (GI No. 164663746).

When nucleic acid encoding the human NIS polypeptide was insertedupstream of the nucleic acid encoding the VSV G polypeptide, functionalvirions were not generated because the NIS expression levels appear tohave been too high for cells to remain viable and allow viralpropagation. Inserting nucleic acid encoding the NIS polypeptidedownstream of the nucleic acid encoding the VSV G polypeptide resultedin the generation of functional NIS-expressing virions due to lowerquantities of NIS polypeptide being produced thereby allowing not onlyefficient viral propagation, but also sufficient quantities of NISpolypeptide for functional iodide uptake in infected cells (FIG. 2B).

Inserting nucleic acid encoding an IFN polypeptide between the nucleicacid encoding the VSV M polypeptide and the nucleic acid encoding theVSV G polypeptide resulted in viruses that infected cells and produced asignificantly increased level of IFN polypeptide expression that wasobserved in the supernatant from infected cells (FIG. 2A). TheVSV-IFN-NIS viruses were able to replicate efficiently in vitro ininfected cells and also express high levels of functional NIS as shownby the ability of the infected cells to take up radio-iodide (FIG. 2B).

Purified stocks of the two VSV-IFN-NIS viruses were titrated on BHK(hamster) cells (FIG. 1B), and cell supernatants were harvested toconfirm the secretion of virally encoded IFNβ. High concentrations ofhuman or murine IFNβ were detected in supernatants of BHK cells infectedwith VSV-hIFN-NIS and VSV-mIFN-NIS, respectively (FIG. 1D), andradioiodine uptake studies confirmed perchlorate sensitive (i.e.,NIS-mediated) concentration of radioactive iodine in virus-infectedcells (FIG. 1C), maximal at 24 hours after high multiplicity infection.

To evaluate the in vivo activity of the VSV-IFN-NIS viruses, the 5TGM1and MPC-11 murine myeloma cell lines were chosen because they reliablyform subcutaneous or orthotopic tumors in immunocompetent syngeneic mice(Lichty et al., Hum. Gene Ther., 15:821-831 (2004) and Turner et al.,Human Gene Therapy, 9:1121-1130 (1998)). Both lines were confirmedsusceptible to VSV-IFN-NIS infection (FIG. 1E), resulting in functionalNIS expression, IFNβ release, and subsequent cell killing. To determinewhether intravenously administered VSV-IFN-NIS viruses could extravasatefrom tumor blood vessels and spread through the parenchyma of the tumor,subcutaneous 5TGM1 or MPC-11 tumors were grown (˜5 mm diameter) insyngeneic mice, a single intravenous dose of 10⁸ TCID50 VSV-IFN-NISvirus was administered, and the biodistribution of virally encoded NISexpression was noninvasively monitored by daily SPECT/CT imaging using99mTcO4 (6 hour half-life) as tracer (FIG. 3A). These tracer uptakestudies indicated that the virus was efficiently extravasating fromtumor blood vessels and suggested that it may be rapidly spreading inthe subcutaneous tumors.

To confirm that the virus was actually replicating and spreading in thetumor parenchyma, selected tumors were harvested immediately afterSPECT/CT imaging at 24, 48, and 72 hours post VSV-IFN-NIS virusadministration and subjected to (i) autoradiography to detect viableNIS-expressing tumor cells; (ii) immunofluorescence (IF) to detect VSVantigens, and (iii) TUNEL staining to identify dead or dying cells.Careful analysis of the data shown in FIG. 3B indicated the existence oflarge, approximately spherical zones of VSV infection in which the tumorcells at the center were apoptotic and those at the periphery remainedviable (see also FIG. 8), express NIS (FIG. 5), and concentrate 99mTcO4(FIG. 9). Quantitative analysis of IF and TUNEL data indicated asignificant increase in the number of virus-infected and apoptotic cellsbetween 24 and 48 hours post virus administration (FIG. 3C). By 72 hoursafter infection, the growing zones of VSV infection largely coalesced,resulting in wholesale tumor destruction (FIG. 3B and FIG. 8).

Additional experiments were conducted to characterize the kinetics ofvirus spread at very early time-points, during the first 24 hours aftervirus administration (FIG. 4A). Analysis of tumor sections harvested 6hours after IV virus administration and stained for both VSV andCD31-positive blood vessels revealed individual scattered VSV infectedcells, mostly close to tumor blood vessels. By 12 hours, small clustersof virus infected cells were visible and by 18 hours they grewsignificantly until by 24 hours they exhibited the typical appearancedescribed previously—apoptotic at the center and viable at theperiphery. Analysis of these dual CD31/VSV stained sections (FIG. 4A,panels i-vi) indicated that the endothelial cells lining tumor bloodvessels did not succumb to VSV infection, even when completelysurrounded by VSV-infected tumor cells, shown at high magnification inFIG. 4B. By plotting average diameters of infectious centers (measuredas number of cells across) at 6, 12, 18, and 24 hour time-points (FIG.4C), it appeared that the virus spread centrifugally at a constant rate,taking approximately 2 hours to infect each successive layer of cells inthe expanding sphere. The rate of accrual of new cells into eachinfectious center therefore increased as the infection progressed, andit was estimated that each center contained approximately 10,000 cellsby 24 hours after virus delivery.

To determine the approximate time from infection to death of infectedtumor cells in vivo, the average diameter of the rim of viable,VSV-infected (i.e. TUNEL-negative, VSV-positive) cells at the advancingedge of intratumoral infection was measured to be approximately 10 cells(FIG. 4D). Thus, the virus spread centrifugally, and it tookapproximately 2 hours to pass the virus on to an adjacent cell (FIG.4C), while cell death did not occur until the rim of infected cellsadvanced by 10 cell diameters (FIG. 4D). Combining these observations,it was concluded that it takes approximately 20 hours for an infectedcells to become apoptotic.

To determine whether efficient extravasation and rapid intratumoralspread of the virus is associated with tumor regression, additionalgroups of C57KaLwRij mice with subcutaneous 5TGM1 tumors were treatedwith a single intravenous dose of 10⁸ TCID50 VSV-IFN-NIS and werefollowed longer term with daily health status checks and tumormeasurements. Tumors regressed rapidly in the majority of VSV-mIFN-NISand VSV-hIFN-NIS treated animals (FIG. 6A). Occasional very early deathswere not associated with neurotoxicity and were presumed due to rapidtumor lysis syndrome, although this was not formally proven.Interestingly, two to three weeks after administration of the viraltherapy, tumor recurrence was seen in most of the animals treated withVSV-hIFN-NIS, but not in those treated with VSV-mIFN-NIS (FIGS. 6A and6B), suggesting that the virally encoded mouse IFNβ, but not the humanIFNβ, was capable of activating mechanisms that lead to the completeeradication of residual disease in this syngeneic immunocompetent mousemodel. Retreatment of relapsing tumors with VSV-IFN-NIS was notattempted since all of the mice had by that time developed high titersof anti-VSV antibodies (FIG. 6C).

Measurement of serum IFNβ levels in virus treated animals indicated thatthis virally encoded cytokine was released into the bloodstream byvirally infected tumor cells at early time-points after virusadministration (FIG. 7A). Antitumor actions of interferon beta includethe direct inhibition of tumor cell proliferation, natural killer cellactivation, anti-angiogenesis, and the enhancement of antitumor T cellresponses. However, proliferation of 5TGM1 and MPC11 myeloma cells invitro was not adversely affected even at high concentrations of IFNβ(FIG. 1F). Moreover, analysis of CD31 or CD3 stained sections of virustreated tumors did not reveal any evidence for inhibition ofanti-angiogenic activity, nor for tumor infiltration by host Tlymphocytes (FIG. 4B). However, virus treated animals whose tumors didnot recur were found to be resistant to re-challenge with 5TGM1 tumorcells (FIG. 7B), indicating that mice had developed 5TGM1 specificantitumor immunity. To determine whether syngeneic VSV-infected myelomacells could provoke a specific anti-myeloma immune response, syngeneicmice were immunized with a single subcutaneous injection of 10⁷VSV-infected 5TGM1 cells, either one day after or five days prior tosubcutaneous tumor cell implantation. Tumor growth was delayed resultingin a significant enhancement of survival in mice that were immunized 5days prior to tumor challenge (FIG. 7C), indicating that theVSV-infected tumor cells provoked a modest antitumor immune response.However, the VSV-infected tumor cell vaccine exhibited no detectableantitumor activity in mice bearing even small, established tumors,suggesting that antitumor immunity was effective only in the context ofminimal disease burden.

To determine whether the lower tumor relapse rates in VSV-mIFN-NIStreated mice could be attributed to virally encoded IFNβ enhancing theantitumor T-cell response, a cocktail of anti-CD4 and anti-CD8antibodies was used to deplete T-cells. Tumors responded equally well tothe intravenous VSV-mIFN-NIS therapy regardless of T cell depletionstatus, but the rate of tumor recurrence was significantly higher inT-cell depleted mice (FIGS. 7D and 7E). These results indicate thateradication of residual tumor cells after oncolytic debulking byVSV-mIFN-NIS was mediated by tumor-specific T cells whose amplificationwas stimulated by the virally encoded mouse IFNβ.

When compared to the VSV-451-NIS virus described in the Goel et al.reference (Blood, 110(7):2342-50 (2007)), which exhibited weak oncolyticefficacy in the immune competent 5TGM1 syngeneic multiple myeloma mousemodel (C57Bl/KalwRijHsd), the VSV-IFN and VSV-IFN-NIS viruses exhibitedgreatly superior replication kinetics. In addition, compared to theVSV-Δ51-NIS virus, the VSV-IFN-NIS viruses induced higher NISpolypeptide expression in vitro. In vivo therapy studies demonstratedthat a single intravenous dose of each of the VSV-IFN and VSV-IFN-NISviruses promoted tumor regression and significantly prolonged survivalof immunocompetent mice bearing subcutaneous or orthotopic 5TGM1 myelomatumors. Tc-99m imaging studies conducted in mice treated withVSV-IFN-NIS viruses exhibited tumor specific viral NIS polypeptideexpression and radio-isotope uptake that increased concurrently withintratumoral viral spread. Further, there were no indications ofneurotoxicity following treatment with the VSV-IFN and VSV-IFN-NISviruses. These results indicate that VSV-IFN-NIS viruses can be used asa therapeutic agent for cancer (e.g., multiple myeloma) that can becombined with radio-isotopes for both non-invasive imaging of viralbiodistribution and radiovirotherapy.

The results provided herein demonstrate that vesicular stomatitisviruses encoding human IFNβ and human NIS exhibit oncolytic efficacy invivo in an immune competent mouse model of multiple myeloma.Systemically administered virus was able to replicate in the tumor,express sufficient levels of functional NIS polypeptides, exert anoncolytic activity to induce tumor regression and improve survival, andexhibit superior NIS expression and oncolytic activity as compared toVSV 451-NIS virus.

Cell Culture and Viruses

Cell lines were cultured in media supplemented with 10% fetal bovineserum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin. BHK-21 andMPC-11 cells, obtained from American Type cell culture (ATCC), weregrown in Dulbecco Modified eagles medium (DMEM). 5TGM1 cells wereobtained from Dr. Babatunde Oyajobi (UT Health Sciences Center, SanAntonio, Tex.) and grown in Iscove's modified Dulbecco medium (IMDM).B-16 murine melanoma cells were obtained from R. Vile and grown in DMEM.All cell lines tested negative for mycoplasma contamination.

Restriction sites were engineered into a pVSV-XN2 plasmid, containingthe VSV positive strand antigenome, at the M/G and the G/L genejunctions preceded by the putative VSV intergenic sequence(TATG(A)₇CTAACAG) required for functional transgene expression (Schnellet al., J. Virol., 70:2318-2323 (1996)). Restriction site flanked cDNAcoding for murine IFNβ, human IFNβ, and NIS genes were generated by PCR.Murine or human IFNβ were incorporated into a single NotI site (M/Gjunction), while NIS was incorporated into XhoI and NheI sites (G/Ljunction) to generate VSV—IFN-NIS plasmid. VSV-IFN-NIS virus was rescuedusing methods described elsewhere (Whelan et al., Proc. Natl. Acad. Sci.USA, 92:8388-8392 (1995)). Viruses were subsequently amplified in BHK-21cells, purified by filtration of cell supernatant, and pelleted bycentrifugation through 10% w/v sucrose. Viral titer was measured inBHK-21 cells following infection using serially diluted virus stock tomeasure Tissue culture infective dose (TCID50) determined using theSpearman and Karber equation.

In Vitro Viral Characterization

Viral titer was measured in supernatant following infection of BHK-21cells (MOI 1.0, 1 hour at 37° C.). To measure in vitro radio-iodideuptake, cells were incubated in Hanks buffered salt solution (HBSS) with10 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, pH7.3) in the presence of radio-labeled NaI (I¹²⁵ at 1×10⁵ cpm)+/−100 μMpotassium perchlorate (KClO₄). IFNβ secretion in supernatant of infectedcells was determined using an enzyme-linked immuno adsorbent assay(ELISA) against murine or human IFNβ (PBL Interferonsource). To compareIFN responsiveness, cells were pre-incubated with 100 U/mL murine IFNβfor 12 hours, followed by infection with VSV-GFP. Proliferation ofviable cells was assessed by MTT assay (ATCC). Killing of 5TGM1 andMPC-11 by VSV-IFN-NIS (MOI 1.0) was similarly monitored at specific timepoints following infection by MTT assay shown as a percentage ofuntreated cells.

In Vivo Studies

5×10⁶ 5 TGM1 or MPC-11 murine myeloma cells were subcutaneouslyimplanted on the right flank of 6-10 week-old syngeneic femaleC57Bl6/KaLwRij (Harlan, Netherlands) or Balb/c mice (Taconic),respectively. Tumor burden was measured by serial caliper measurements.Mice were administered with a single, intravenous dose of 1×10⁸/0.1 mLVSV-IFN-NIS or equal volume PBS by tail vein injection. SPECT-CT imagingwas carried out following intraperitoneal (IP) administration of 0.5 mCiTc-99m and quantified as described elsewhere (Penheiter et al., AIR Am.J Roentgenol., 195:341-349 (2010)).

High Resolution Tumor Analysis

Tumors harvested at 24 hour intervals were frozen in OCT for sectioning.Tumor sections were analyzed by autoradiography and immunofluorescence(IF) for (i) VSV antigens using polyclonal rabbit anti-VSV generatedin-house in the viral vector production labs at the Mayo Clinic,followed by Alexa-labeled anti-rabbit IgG secondary antibody(Invitrogen, Molecular Probes), (ii) cell death by TUNEL staining(DeadEnd™ Fluorometric TUNEL kit, Promega), and (iii) cellular nucleiusing Hoescht 33342 (Invitrogen). Image quantification was performed onfour random images from n=3 VSV-mIFN-NIS treated tumors (except n=2tumors at 72 hours post treatment) using ImageJ software to obtain VSVor TUNEL(+) regions as percentage of tumor area. IF analysis of tumorsharvested at 6 hour intervals detected VSV antigens and tumor bloodvessels using a rat anti-mouse CD31 antibody (BD Pharmingen).Intratumoral foci size was quantified by measuring 7-8 foci from 2tumors and dividing diameter by average tumor cell size (based ondiameter measurements of 50 individual cells) to obtain foci diameter innumbers of cells. Volume of approximately spherical foci was estimatedusing formula, v=4/3(π*r3). Average width of rim of viable, VSV-infectedcells was similarly quantified from IF images from n=3 tumors harvestedat 48 hours post VSV—IFN-NIS administration.

Immune Studies in Immune Competent Mice

To measure generation of antiviral antibodies, serial 2-fold dilutionsof heat-inactivated serum were pre-incubated with 500 TCTID₅₀ VSV-GFP,and subsequently used to infect BHK-21 cells. Minimum serum dilutionallowing VSV induced CPE was plotted. In vivo IFNβ secretion wasmeasured in serum by ELISA. 5TGM1 vaccinations were administered byinjecting 1×10⁷ VSV-mIFN-NIS infected cells (MOI 10.0) subcutaneously inthe left flank of syngeneic mice. T-cell depletion studies wereperformed in C57Bl6/KaLwRij mice by intraperitoneal administration ofanti-CD4 and anti-CD8 antibodies (50 μg each) administered 3 times/week,followed by a weekly maintenance dose.

Statistical Methods

Visual displays of the data were used to assess for outliers orsubstantial departures from normality, and t-test was utilized wheredescribed. In all cases, two-tail P-values were provided which are notadjusted for multiple comparisons. Comparison of survival differenceswas performed using Log-rank test from Kaplan meier survival curves. Forcomparing tumor relapse rates in animal studies, the Fischer exact testwas utilized due to small sample size.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for treating cancer, wherein said method comprisesintravenously administering a composition comprising vesicularstomatitis viruses to a mammal comprising cancer cells at a dose fromabout 10³ pfu to about 10¹² pfu, wherein said vesicular stomatitisviruses comprise an RNA molecule comprising, in a 3′ to 5′ direction, anucleic acid sequence that is a template for a positive sense transcriptencoding a VSV N polypeptide, a nucleic acid sequence that is a templatefor a positive sense transcript encoding a VSV P polypeptide, a nucleicacid sequence that is a template for a positive sense transcriptencoding a VSV M polypeptide, a nucleic acid sequence that is a templatefor a positive sense transcript encoding an IFN polypeptide, a nucleicacid sequence that is a template for a positive sense transcriptencoding a VSV G polypeptide, a nucleic acid sequence that is a templatefor a positive sense transcript encoding a NIS polypeptide, and anucleic acid sequence that is a template for a positive sense transcriptencoding a VSV L polypeptide, wherein administration of said compositionto said mammal is under conditions wherein said vesicular stomatitisviruses infect said cancer cells to form infected cancer cells, whereinsaid infected cancer cells express said IFN polypeptide and said NISpolypeptide, and wherein the number of cancer cells within said mammalis reduced following said administration.
 2. The method of claim 1,wherein said mammal is a human.
 3. The method of claim 1, wherein saidIFN polypeptide is a human IFN beta polypeptide.
 4. The method of claim1, wherein said NIS polypeptide is a human NIS polypeptide.
 5. A methodfor inducing tumor regression in a mammal, wherein said method comprisesintravenously administering a composition comprising vesicularstomatitis viruses to a mammal comprising a tumor at a dose from about10³ pfu to about 10¹² fu wherein said vesicular stomatitis virusescomprise an RNA molecule comprising, in a 3′ to 5′ direction, a nucleicacid sequence that is a template for a positive sense transcriptencoding a VSV N polypeptide, a nucleic acid sequence that is a templatefor a positive sense transcript encoding a VSV P polypeptide, a nucleicacid sequence that is a template for a positive sense transcriptencoding a VSV M polypeptide, a nucleic acid sequence that is a templatefor a positive sense transcript encoding an IFN polypeptide, a nucleicacid sequence that is a template for a positive sense transcriptencoding a VSV G polypeptide, a nucleic acid sequence that is a templatefor a positive sense transcript encoding a NIS polypeptide, and anucleic acid sequence that is a template for a positive sense transcriptencoding a VSV L polypeptide, wherein administration of said compositionto said mammal is under conditions wherein said vesicular stomatitisviruses infect tumor cells of said tumor to form infected tumor cells,wherein said infected tumor cells express said IFN polypeptide and saidNIS polypeptide.
 6. The method of claim 5, wherein said mammal is ahuman.
 7. The method of claim 5, wherein said IFN polypeptide is a humanIFN beta polypeptide.
 8. The method of claim 5, wherein said NISpolypeptide is a human NIS polypeptide.